An optimized yeast display strategy for efficient scFv antibody selection using ribosomal skipping system and thermo resistant yeast.

OBJECTIVES: Establish a method to restrict unexpected fragments including stop codons in scFv library and generate a thermo resistant strain for screening of thermal stable scFv sequences. RESULTS: Here, we have constructed a T2A-Leu2 system for selection of yeast surface display libraries that blocks amplification of "stop codon" plasmids within the library, thereby increasing the quality of the library and efficiency of the selection screen. Also, we generated a temperature-resistant yeast strain, TR1, and validated its combined use with T2A-Leu2 for efficient screening. Thus, we developed a general approach for a fast and efficient screening of scFv libraries using a ribosomal skipping system and thermo-resistant yeast. CONCLUSIONS: The method highlights the utility of the T2A-Leu2-based ribosomal skipping strategy for increasing the quality of the input library for selection, along with an optimized selection protocol based on thermo-resistant yeast cells.

The Effect of Polyhydroxy Fullerene Derivative on Human Myeloid Leukemia K562 Cells.

The use of nanomedicines for cancer treatment has been widespread. Fullerenes have significant effects in the treatment of solid tumors. Here, we are going to study the effects of hydroxylated fullerene C60(OH)n(n = 18-22) treatment on chronic myeloid leukemia cell proliferation and investigate its toxicity. The results showed that hydroxylated fullerene C60(OH)n (n = 18-22) at low concentrations (less than 120 µM) not only had apparent toxic side effects, but also promoted the growth of K562 cells, while a high concentration of C60(OH)n had different degrees of inhibition on K562 cells. When the concentration is higher than 160 µM, the K562 cells showed morphological changes, the mitochondrial membrane potential decreased, the cell cycle was blocked in the stage of G2-phase, and cell apoptosis occurred, which may cause apoptosis, autophagy, and a variety of other damage leading to cell death. Meanwhile, it also indicated that its inhibition of solid tumors might be related to the tumor microenvironment; we verified the safety of fullerene without apparent cellular toxicity at a specific concentration.

Deep insight into reasons for the mechanoluminescence phenomenon of triphenylamine-substituted imidazoles and their distinct mechano-fluorochromic behaviours.

Three imidazoles with different numbers of fused aromatic rings have been prepared by the respective introduction of triphenylamine and 4-cyanophenyl at the N1 and C2 positions in the imidazole ring. Each imidazole effectively exhibits positive solvatochromism, and that of benzo[d]imidazole is the most significant. Although these imidazole crystals have centrosymmetric space groups, they are all ML-active. It was verified by DFT calculations based on X-ray crystallography that some molecular couples with strong intermolecular interactions possess large net dipole moments that should be dominantly responsible for the ML behaviours of these crystals. Moreover, the considerably high molecular dipole moments of the three imidazoles also make a great contribution to good ML effects. Based on this triphenylamine-substituted imidazole system, the relationships among space groups, molecular dipole moments, polar molecular couples and the ML phenomenon are made clear for the first time. Unlike the remarkable MFC activities on imidazole and benzo[d]midazole crystals, phenanthro[9,10-d]imidazole is MFC-inert, and this may well be attributed to strong intramolecular C-H⋯π interactions, which make the rotation of triphenylamine nearly impossible under force stimuli.

Dual Repressive Function by Cip1, a Budding Yeast Analog of p21, in Cell-Cycle START Regulation.

Cip1, a newly identified yeast analog of p21, is a Cln3-CDK inhibitor that negatively regulates cell-cycle START. However, its function remains poorly understood. In this study, we found that deletion of CLN3 did not result in bypass of G1-phase arrest caused by Cip1 overexpression. Cip1 depletion in cln3-null mutants significantly advanced the timing of Cln2 expression, supporting the idea that Cip1 represses START in a Cln3-independent manner. We set to search for novel Cip1 interacting proteins and found that Ccr4, a known START regulator, and its associated factor Caf120, interact with Cip1. Ccr4-Caf120 acts redundantly with Cdk1-Cln3 to inhibit Whi5-mediated regulation of START. This interaction was conserved between human Ccr4 and p21. In addition, deletion of WHI5 robustly suppressed G1-phase arrest caused by Cip1 overexpression. We conclude that Cip1 negatively regulates START by acting as a dual repressor of Ccr4 in parallel with Cln3.

A new strategy for seamless gene editing and marker recycling in Saccharomyces cerevisiae using lethal effect of Cwp1.

Technologies development for seamless gene editing and marker recycling has allowed frequent genomic engineering in Saccharomyces cerevisiae for desired laboratory strains and cell factory. Alternative new approaches are still required for complicated scenarios. In this study, we report that inducible overexpression of cell wall protein 1 (Cwp1) by galactose addition confers yeast cells a robust growth inhibition. Direct repeats flanking the Gal-CWP1:selectable marker cassette allow for its homology recombination excision and counter selection upon galactose addition, therefore enable seamless gene editing and marker recycling. We used this strategy and efficiently generated scarless Ade8 deletion mutants. Our results highlight the utility of lethal effect of Cwp1 overexpression a new counter selection strategy and a simple and efficient method for seamless gene editing and marker recycling in S. cerevisiae and potentially other fungi.

Ordered Coimmobilization of a Multienzyme Cascade System with a Metal Organic Framework in a Membrane: Reduction of CO2 to Methanol.

Enzymatic reduction of CO2 is of great significant, which involves an efficient multienzyme cascade system (MECS). In this work, formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), and reduced pyridine nucleotide (NADH) (FDH&GDH&NADH), formaldehyde dehydrogenase (FalDH), GDH, and NADH (FalDH&GDH&NADH), and alcohol dehydrogenase (ADH), GDH, and NADH (ADH&GDH&NADH) were embedded in ZIF-8 (one kind of metal organic framework) to prepare three kinds of enzymes and coenzymes/ZIF-8 nanocomposites. Then by dead-end filtration these nanocomposites were sequentially located in a microporous membrane, which was combined with a pervaporation membrane to timely achieve the separation of product methanol. Incorporation of the pervaporation membrane was helpful to control reaction direction, and the methanol amount increased from 5.8 ± 0.5 to 6.7 ± 0.8 µmol. The reaction efficiency of an immobilized enzymes-ordered distribution in a membrane was higher than that disordered distribution in the membrane, and the methanol amount increased from 6.7 ± 0.8 to 12.6 ± 0.6 µmol. Moreover, it appeared that introduction of NADH into ZIF-8 enhanced the transformation of CO2 to methanol from 12.6 ± 0.6 to 13.4 ± 0.9 µmol. Over 50% of their original productivity was retained after 12 h of use. This method has wide applicability and can be used in other kinds of multienzyme systems.